As a result of the rapidly developing genomic testing, whole nucleic acid analysis is an increasing task in many genetic laboratories. The polymerase chain reaction (PCR) is a well established method for amplifying nucleic acid sequences, and the method is routinely used in numerous application areas, such as microbiological testing, expression studies, determination of genetic variation in population, and genetic testing, forensics and food and environmental testing. Testing of nucleic acids using PCR generally involves three steps: sample preparation, amplification and detection. However, the processes for performing the tests used today are often laborious. The current trend is towards simplified assays allowing automation for nucleic acid analysis.
The increased number of tests to be run raises a need for cost-effective operations based on integration and automation of the assay procedures, and the automation of the whole DNA analysis is an increasing task in many genetic laboratories. Although several attempts to perform DNA-analysis as high throughput assays has been described, the logistics of sample handling from sample preparation and pre-treatment to the ultimate analysis still requires manual handling and physical transportation of the samples. In addition, the need to physically transfer amplified DNA samples within the lab poses a serious contamination risk.
One type of a simplified assay that reduces sample handling is provided by so-called closed-tube assays. In closed-tube assays, the FOR product is analyzed in the amplification tube by a homogeneous method, such as TaqMan® (U.S. Pat. No. 5,210,015) or by molecular beacons (U.S. Pat. No. 5,118,801). These approaches allow integration of amplification and detection. However, they lack integration of sample preparation with amplification and detection.
Pre-treatment of the sample is required to remove common inhibitors to nucleic acid amplification that may be present in samples from biological sources. Inhibitors to amplification include, e.g. naturally occurring chelating agents, enzymes and/or proteins that can damage either nucleic acid templates or PCR polymerases used in the amplification reactions. In addition, the common anticoagulants that are used to treat whole blood samples can interfere with nucleic acid amplification reactions.
Numerous technologies have been developed to purify nucleic acids from biological samples but all available procedures are time-consuming and labour intensive. There are several automated stations for sample preparation available on the market, based on silica-chaotrop extraction (U.S. Pat. No. 5,234,809) columns, such as QIAamp®, or the like, and various magnetic bead systems. A different approach is the FTA® Technology (U.S. Pat. No. 5,496,562) that lyses cell membranes as soon as the sample is applied onto a coated filtration matrix allowing immobilization of nucleic acids onto the matrix. After washing, the nucleic acids can be released in a manner that enables them to be amplified by PCR.
Another approach is represented by the development of different solid matrices for collecting, transporting, storing and purifying biological samples, such as clinical whole blood, saliva or faecal samples, for nucleic acid analysis. U.S. Pat. No. 5,807,527 describes a solid medium for long term storage of blood DNA, which comprises a composition, which protects against degradation of DNA, a protein denaturing agent and a free radical trap.
Another example of this approach is the described in U.S. Pat. No. 5,939,259, which discloses an absorbent material, which does not bind nucleic acids irreversibly which is impregnated with a chaotropic salt.
In these known technologies, the DNA of stored blood samples is extracted from the medium before performing PCR, or the DNA is used in PCR in situ on the solid medium after extensive purification, as described in U.S. Pat. No. 5,807,527. Extraction or elution of DNA from the medium requires a multi-step procedure with special solutions and incubations.
Known technologies using solid media for collecting, storing and purifying DNA thus involve multi-step procedures, some using several separate vials and solutions, for generating a sample useful for amplification and detection. Moreover, certain of these methods unnecessarily produce waste.